PROJECT SUMMARY Neuromuscular junctions (NMJs), a unique synaptic site where spinal motor neurons (MNs) meet myofibers to form a functional motor unit (MU), consist of three major components, MN?s pre-synapse, myofiber?s post- synapse, and terminal Schwann cells (SCs). Increasing attention has recently been paid to the etiology of ?NMJ aging? in relationship to sarcopenia, the loss of muscle mass and strength associated with aging. Here, we propose two complementary aims focusing on unraveling NMJ aging mechanisms in relation to sarcopenia: <SA-1> Identifying the earliest sarcopenia-associated molecular changes and potential biomarkers at aging NMJs in vivo - We will perform, for the first time, RNAseq of micro-dissected NMJs, isolated at different time points from aging mice, using a laser-capture micro-dissection (LCM) technique. A series of established bioinformatics analyses will be then run to extract candidate molecules followed by identification of their cellular origins. To obtain high-quality RNA in the LCM, we will apply our original technique, which requires only a few minutes to visualize the NMJ. We will prioritize the validation of therapeutic target candidates previously associated with neuromuscular diseases and biomarker candidates with known ligands or other potential monitoring strategies. <SA-II> Engineering a novel in vitro co-culture system of mature and aging NMJs - We will employ a double-compartmented system recapitulating the physiological separation of the central (MNs and astrocytes [ASTs]) and peripheral (MN axons, myotubes [MTs] and SCs) nervous systems in vivo. The glial cells (ASTs and SCs) will provide the MU with the biologically required structural and trophic support, minimizing the need for culture media supplementation. This strategy is essential for a faithful subsequent modeling of NMJ aging, which is thought to be associated with functional decline of cells constituting the MUs. In addition, we will protect cells in both compartments with fibrin gel scaffoldings to prevent cell detachment and synaptic disconnection due to frequent MT contraction. Furthermore, intermittent electrical stimulation (IES) will be provided to the MUs to facilitate their maturation. Once maturity is achieved, we will terminate the ?muscle exercise effect? of IES and reduce trophic factors to mimic natural aging in humans. NMJ maturity and aging will be monitored at multiple time points for molecular, morphological, and functional changes. Finally, the co-culture system developed will be utilized for screening and determining the properties of the molecules identified in SA-I. <Study impact> Our novel NMJ-LCM with a cutting-edge bioinformatics analysis should generate invaluable data to reveal the pathology and gain potential therapeutic insights into sarcopenia. In addition, our innovative co-culture system could become the first in vitro model to achieve complete maturation and subsequent aging of NMJs and serve as a powerful tool for mechanistic and translational studies of sarcopenia. This work will also contribute to a broader area of neuromuscular research, ranging from basic studies on MUs/NMJs, to more translational works on various other neuromuscular diseases.